1. Field of the Invention
The present invention relates to an apparatus for total internal reflection microscopy having an objective lens and a coupling element for illuminating the specimen through the objective lens in a fashion that allows evanescent field-illumination, epi-illumination or both.
2. Description of Related Art
The principle of total internal reflection (TIR), which prevents a light beam exceeding a given angle of incidence from leaving a medium having a higher refractive index into a medium having a lower refractive index is increasingly utilized for epi-fluorescence microscopy (“total internal reflection fluorescence (TIRF)”). Therein the fact is utilized that the electromagnetic field of the totally reflected light beam extends into the medium having the lower refractive index and is able to excite fluorescent molecules there. The penetration depth of this so-called evanescent field depends on the wavelength “λ” and the angle of reflection “α” and usually is about several hundred nanometers. Thus it is possible to distinguish fluorophores located close to the reflecting interface from those located further away from it. An angle sufficiently large for total reflection within the substrate can either be achieved by coupling the excitation light beam laterally into the support substrate, or by utilizing special immersion objective lenses having an extraordinarily high numerical aperture. Such objectives allow to focus light onto the specimen at an angle exceeding the threshold angle of total internal reflection.
Lasers are usually used as light sources for TIR epi-fluorescence. A diffraction limited laser focus is projected into the rear focal plane of an appropriate objective lens having a sufficiently high numerical aperture. Upon passing through the objective lens the laser light is collimated, whereby the exact focus position in the back focal plane (pupil) of the objective lens determines the angle of incidence of the beam of light on the sample according to equation 1:sinα=r/(n0f)
The condition for total internal reflection, on the other hand, is given by equation 2:n0sinα=n1wherein                r: distance of the laser focus from the optical axis;        n1: refractive index of the specimen medium;        n0: refractive index of the substrate or the immersion medium;        f: focal distance of the objective lens        
For positioning the laser focus in the desired focal position within the pupil of the objective lens the laser light usually is coupled into the beam by means of a beam splitter element. However, if the laser beam does not hit the beam splitter at the right angle or if the beamsplitter is not tilted correctly, laser light may enter areas of the pupil of the objective lens, which do not result in total reflection.
The use of a laser as a preferred light source for TIR epi-fluorescence is a consequence of the low illumination depth of the TIR arrangement. Usually only a few fluorophores are excited in the narrow evanescent field, hence the resulting signals are usually very weak. If it is not possible to increase the sensitivity of the detector, the excitation energy has to be increased for achieving a good signal to noise ratio. On the other hand, when utilizing sufficiently strong lasers, even minute changes of the adjustment of the laser beam may result in a laser beam, which doesn't undergo total internal reflection. Instead it may pass through the specimen and reach the experimenter's eye where it may cause significant and harmful damage. U.S. patent application Ser. No. 2002/0097489 A1 discloses a microscope system for TIR illumination wherein exclusively white light is used, which passes through an annular aperture prior to being coupled into the illumination beam by reflection via a separate beam splitter. This beam splitter serves to combine normal epi-illumination light with TIR illumination light. A drawback of this microscope is its relatively complicated design.
It is an object of the invention to provide for an apparatus for TIR microscopy having a coupling element for TIR illumination and simultaneously for light from another light source for epi-illumination, wherein a reliable protection from faulty operation during adjustment is achieved, wherein the apparatus is also particularly suitable for the use of lasers having a high output power, and wherein the apparatus has a particularly simple design.